Cyclic AMP is a major second messenger playing an important role in electrolyte transport in intestinal and renal epithelial cells and contributing to pathophysiology of diarrheal diseases. An increased cAMP level in intestine is a major cause of diarrheal diseases primarily by increasing C1- secretion by crypt cells and by decreasing Na+ absorption by villus cells. The decrease in Na+ absorption in villus cells by cAMP is poorly understood except that inhibition of brush border Na+/H+ exchanger, NHE3, in enterocytes is a target. We were surprised that cAMP failed to regulate the brush border Na+/H+ exchanger, NHE3, in two model system, PS120 fibroblasts and Caco-2 cells. Using a yeast two-hybrid system, we have identified two regulatory proteins, E3KARP and NHERF, which are necessary for NHE3 inhibition by cAMP. These proteins show a limited homology but have a similar human tissue distribution. PS120 fibroblasts and human colonic carcinoma Caco-2 cell line lack endogenous expression of these regulatory proteins. When these regulatory proteins are expressed in PS120 fibroblasts stably transfected with NHE3, both regulatory proteins can reconstitute the cAMP-induced inhibition of NHE3, suggesting the necessity of the regulatory proteins. In order to understand the molecular basis for NHE3 inhabitation by cAMP, we propose to investigate interactions between NHE3 and the regulatory proteins at a molecular level. (1) We will investigate the effect of cAMP on changes in phosphorylation of NHE3 and the regulatory proteins in Caco-2 cells. (2) How the regulatory proteins transduce cAMP effects on NHE3 is not known. We hypothesize that the regulatory proteins may function as scaffold proteins for proteins kinase A, assembling components involved in cellular signaling at the plasma membrane. We will study interaction between PKA and the regulatory proteins. (3) We will raise antibodies against the regulatory proteins and determine subcellular localization of the regulatory proteins and NHE3. (4) If E3KARP and NHER are expressed in the same cells, we will investigate potential interactions between the two regulatory proteins such as dimerization and synergy in NHE3 inhibition by cAMP. (5) We will also determine the interaction domains within NHE3 and the regulatory proteins using a yeast two-hybrid system.